During the course of the previous project, our understanding of the organization of the mammalian circadian system changed considerably as a result of evidence indicating that many peripheral tissues in vivo and fibroblast cell lines in vitro also express oscillations in molecular components of the canonical clockworks. Yet, despite these oscillatory properties, peripheral tissues and fibroblast cell lines cannot function as pacemakers by regulating circadian rhythms in other cells or downstream processes. Only oscillators derived from the suprachiasmatic nucleus (SCN)possess the capability to restore behavioral rhythmicity when transplanted into SCN-lesioned hosts, and to coordinate molecular and physiological oscillations in co- cultured cells. The pacemaker function of SCN oscillators is presumably derived from the distinctive nature of their outputs that mediate cellular communication between cell-autonomous clocks within the SCN and from the SCN to downstream oscillators. Using immortalized rat SCN cells (SCN2.2), we have shown that pacemaker regulation of circadian rhythms in other co-cultured cells is mediated by SCN-specific diffusible factors. Therefore, the long-term objective of this project is to identify the diffusible outputs that communicate rhythmicity to co-cultures of other cell types in vitro and restore circadian wheel-running behavior when transplanted into arrhythmic hosts in vivo. Experiments will use multi-faceted approaches to determine whether: 1) antisense/siRNA and/or pharmacological inhibition of candidates for circadian diffusible signals in SCN2.2 cells or their receptor/response elements in co-cultured cells disrupts rhythmicity in these downstream cells; 2) clonal lines of immortalized SCN cells that express a specific output signal are capable of generating endogenous oscillations and coordinating rhythms in co-cultured cells; and 3) clonal SCN cell lines with pacemaker properties in vitro also confer behavioral rhythmicity to SCN-lesioned rats and transgenic mice with mutant circadian phenotypes. These studies will yield novel information on how SCN circadian outputs coordinate oscillations in different downstream tissues and cells, and what cellular processes distinguish the function of SCN oscillators as a circadian pacemaker. Lay Summary: Such information will lead to new developments in the understanding, diagnosis and treatment of disorders in human health and performance that may result from internal desynchronization of body processes such as depression, sleep disturbances, diabetes, obesity and carcinogenesis.